microRNA文库构建及其高通量测序和定量分析

The microRNA (miRNA) related studies are one of the fastest growing frontier research areas. The analytical methods for miRNA detection are pivotal in the development of miRNA-related studies. Although the analytical methods have been recently developed in rapid progress, the miRNA analysis still has some bottleneck problems to be solved, including highly sensitive detection of 2-O-methylated plant miRNAs, high throughput analysis methods universally suitable to detection of plant and animal miRNAs and high throughput sequencing of miRNAs. Through creative design of stem-loop DNA probes, this project wants to systematically solve the bottleneck problems of miRNA analysis. Firstly, based on the rapid and efficient loop-mediated isothermal amplification initiated by the reverse transcription and extension of the stem-loop DNA probes, we will study and establish the highly sensitive analysis methods for detection of plant miRNAs. Secondly, through the reverse transcription of the stem-loop DNA probes containing random bases, we will efficiently construct the cDNA library of miRNAs and realize the high-throughput analysis methods for quantitative detection of animal and plant miRNAs. Finally, through the reverse transcription and extension of a pair of stem-loop DNA probes, we will efficiently construct the cDNA library of miRNAs suitable to high-throughput miRNA sequencing and subsequently realize the high-throughput miRNA sequencing. The studies of this project will provide a powerful analysis technologies for miRNA-related studies of molecular biology and miRNA-based clinical diagnosis.

有关microRNA (miRNA)的研究是目前发展最快的前沿领域之一，miRNA的分析在其中起着关键的作用。近年来miRNA的分析研究取得了快速发展，但存在着几个急待解决的关键问题：2-O-甲基化的植物miRNA的分析、miRNA的高通量分析及miRNA的高通量测序。本项目拟通过创新性设计，系统研究解决miRNA分析中的关键问题：（1）通过设计茎环DNA探针的逆转录及延伸反应，引发高效的环介导等温指数扩增反应，实现植物miRNA的高灵敏度分析；（2）通过含有随机碱基的茎环DNA探针逆转录，建立miRNA的cDNA文库，并实现普遍适用于动、植物miRNA的高通量分析；（3）通过一对含有随机碱基的茎环DNA探针的逆转录和延伸反应，建立适合于动、植物miRNA高通量测序的cDNA文库，实现其高通量测序。由此为miRNA的基础研究及其在疾病诊断方面的应用提供强有力的分析技术。

按照申请书提出的研究任务和研究目标，本研究项目以miRNA为研究对象，围绕植物miRNA分析、miRNA高通量分析、miRNA快速可视化分析、miRNA单细胞分析等问题，（1）利用茎环结构DNA探针的逆转录、延伸反应，首次实现了普遍适用于动、植物的均相溶液中单分子、单细胞水平的miRNA高灵敏度分析。（2）研究解决miRNA cDNA文库建立中引物二聚体非特异性扩增的关键问题，实现了高效测序文库构建，并创新性设计了探针长度、荧光波长二维编码策略，实现了一管反应同时检测多种miRNA的高通量分析。（3）利用茎环结构DNA探针研究实现了miRNA cDNA高效文库构建；（4）基于特异性连接酶所催化的DNA探针连接反应，结合PCR技术、环介导等温核酸扩增技术建立一系列高灵敏度、高特异性miRNA定量检测新方法，并将这些方法推广至各类核酸标志物分子（包括环形RNA、mRNA剪接变体、融合mRNA转录本、DNA甲基化、SNP）的特异性性识别及定量分析。（5）除此之外，建立自驱动DNA水凝胶传感器，实现了无需扩增、无需仪器的miRNA可视化、快速分析，为基于核酸分子的POCT诊断分析提供了新思路；研究构建了单微球信号富集和信号放大系统，实现了对外泌体及外泌体中癌症相关蛋白的定量分析，取得了一系列创新性的研究成果。